TIA-1 binding proteins and isolated complementary DNA encoding the same

ABSTRACT

Complementary DNA (cDNA) has been isolated having a sequence that encodes a polypeptide that binds TIA-1 in a double transformation. In one embodiment, the polypeptide is immunologically reactive with the monoclonal antibody produced by the hybridoma designated ATCC #HB-11721. Specific cDNA sequences have been determined and amino acid sequences have been deduced therefrom.

This invention was made with government support under Grant numbers AI33600 and CA 53595 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

This application is a Continuation-in-Part of application Ser. No.08/133,530, filed Oct. 7, 1993 now abandoned.

FIELD OF THE INVENTION

The present invention relates to proteins that bind to TIA-1 and thatare associated with lymphocytes. The present invention also relates toisolated cDNA encoding the binding proteins.

BACKGROUND OF THE INVENTION

Cytolytic lymphocytes (CTLs) possess cytoplasmic granules that arereleased in response to target cell recognition. CTL granules containsecretory proteins such as perforin and serine proteases, which arethought to contribute to the induction of target cell death. Perforinhas been shown to be directly cytolytic. In the presence of Ca⁺⁺, itinserts into the target cell plasma membrane where it aggregates to formosmotically active ion channels {Lichtenheld, M. G., et al, (1988),"Structure and function of human perforin", Nature, 335:448-451; Hameed,A., et al, (1989), "Cytolysis by Ca-permeable transmembrane channels.Pore formation causes extensive DNA-degradation and cell lysis", J. Exp.Med., 169:765-777}. The recent demonstration that transfection ofperforin cDNA into rat basophilic leukemia (RBL) cells confers theability to lyse erythrocytes via a regulated secretory mechanismsupports a direct role for perforin in lymphocyte-mediated cytolysis{Shiver, J. W. and P. A. Henkart, (1991), "A noncytotoxic mast celltumor line exhibits potent IgE-dependent cytotoxicity after transfectionwith the cytolysin/perforin gene", Cell 64:1175-1181}. The inability ofperforin-transfected RBL cells to efficiently lyse nucleated cells,however, suggests that additional granule components are required foroptimal lymphocyte-mediated killing. That perforin is not the onlycytolytic effector molecule is supported by the ability of naturalkiller (NK) cells and CTLs to kill some target cells in the absence ofextracellular Ca⁺⁺, which is required for perforin activity {Tirosh, R.and G. Berke, (1985), "T Lymphocyte mediated cytolysis as an excitatoryprocess of the target. I. Evidence that the target may be the site ofcalcium action", Cell Immunol., 75:113-123}. Furthermore, cytolyticlymphocytes that express little or no perforin (e.g., CD4⁺ CTL clones)have been shown to be potent cytolytic effector cells {Takayama, H., etal, (1991), "Antigen-specific directional target cell lysis byperforin-negative T lymphocyte clones", Inter. Immunol., 3:1149-1156}.The results imply that perforin-independent cytolytic effectormechanisms contribute to at least some forms of target cell killing.

In addition to perforin-mediated lysis, CTLs have been shown to inducein target cells a pathway of programmed cell death known as apoptosis{Russell, J. H. (1983), "Internal disintegration model of cytotoxiclymphocyte-induced target damage", Immunol. Rev., 72:97-118}. Aconvenient marker of this autolytic pathway is the fragmentation oftarget cell DNA into integer multiples of a 200 bp nucleosome-sizedmonomer {Wyllie, A. H., (1980), "Glucocorticoid-induced thymocyteapoptosis is associated with endogenous endonuclease activation", Nature284:555-556; Duke, R. C., et al, (1983), "Endogenousendonuclease-induced DNA fragmentation: an early event in cell-mediatedcytolysis", Proc. Natl. Acad. Sci., 80:6361-6365}. The resulting"ladder" of DNA fragments is considered to be characteristic of thisprogrammed suicide pathway. The observation that perforin induces celllysis, but not DNA fragmentation {Duke, R. C., et al, (1989), "Purifiedperforin induces target cell lysis but not DNA fragmentation", J. Exp.Med., 170:1451-1456} suggests that other granule components are likelyto be responsible for the induction of apoptotic cell death. Thegranzymes, a family of granule-associated serine proteases, arecandidate perforin-independent cytolytic effector molecules {Pasternack,M. S. and H. N. Eisen, (1985), "A novel serine esterase expressed bycytotoxic T lymphocytes", Nature, 314:743-745; Masson, D. and J.Tschopp, (1987), "A family of serine esterases in lytic granules ofcytolytic T lymphocytes", Cell 49:679-685}. Although purified granzymesare not directly cytotoxic, the ability of protease inhibitors to blocklymphocyte-mediated cytolysis suggests that they play a role in targetcell killing {Lavie, G., et al, (1985), "The mechanism of human NK cellmediated cytotoxicity. Mode of action of surface-associated proteases inthe early stages of the lytic reaction", J. Immunol., 135:1470-1476;Rodgers, K. E., et al, (1988), "Inhibition of cytotoxic T lymphocyte andnatural killer cell-mediated lysis by O,S,S-trimethyl phosphorodithioateis at an early post-recognition step", J. Immunol., 140:564-570}. Theobservation that granzyme A, the most abundant granule-associated serineprotease, can induce DNA fragmentation in detergent permeabilized EL4cells argues that these molecules might contribute to the induction ofapoptosis in CTL targets {Hayes, M. P., et al, (1989), "Induction oftarget cell DNA release by the cytotoxic T lymphocyte granule proteasegranzyme A", J. Exp. Med., 170:933-946}. The further demonstration thatthe combination of granzymes and perforin can induce DNA fragmentationin unpermeabilized target cells suggests that perforin might be involvedin the delivery of granzymes to target cells {Hayes, et al, supra,(1989); Shi, L., et al, (1992), "A natural killer cell granule proteinthat induces DNA fragmentation and apoptosis", J. Exp. Med.,175:553-566}. Finally, transfection of RBL cells with a combination ofperforin and granzyme A confers the ability to induce DNA fragmentationin selected target cells {Shiver and Henkart, supra, (1991)}. Becausethe amount of DNA fragmentation induced by these cells is significantlyless than that induced by CTLs, it is possible that additionalgranule-associated molecules are involved in the induction of apoptoticcell death.

Recently, another class of granule-associated proteins that are alsoable to induce DNA fragmentation in CTL target cells has beenidentified. TIA-1 is an RNA-binding protein that was initiallyidentified by a monoclonal antibody (2G9) reactive with a 15 kD proteinwhose expression was restricted to CTLs and NK cells {Anderson, P., etal, (1990), "A monoclonal antibody reactive with a 15-kDa cytoplasmicgranule-associated protein defines a subpopulation of CD8+ Tlymphocytes", J. Immunol., 144:574-582}. Mitogenic activation inducedthe expression of immunoreactive isoforms of TIA-1 that migrated at 28kD, 40 kD and 53 kD. Immunoselection of a λgt11 cDNA library using themonoclonal antibody reactive with TIA-1 identified two related cDNAsthat encode p15-TIA-1 (1T4T8.9-5, 1.6 kb) and p40-TIA-1 (12G9.4, 2.2 kb){Tian, Q., et al, (1991), "A polyadenylate binding protein localized tothe granules of cytolytic lymphocytes induces DNA fragmentation intarget cells", Cell, 67:629-639}. Both TIA-1 isoforms were able toinduce DNA fragmentation in permeabilized target cells, suggesting thatthey might be the granule-associated proteins responsible for theinduction of apoptotic cell death in CTL target cells. Nothing is knownabout the molecular mechanisms by which TIA-1 triggers DNA fragmentationin target cells. Identification of cDNAs encoding TIA-1 binding proteinswould be a first step in the molecular characterization of TIA-1function. Further, characterization of the proteins would be useful toscreen for drugs that induce apoptotic death in target cells.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to identify cDNAsencoding TIA-1 binding proteins.

These and other objects have been achieved by providing isolated cDNAcomprising a sequence that encodes a polypeptide that binds TIA-1 in adouble transformation.

In a preferred embodiment, the isolated cDNA sequence that encodes apolypeptide is SEQ ID NO:1 or SEQ ID NO:3.

The invention further provides isolated cDNA that hybridizes understringent conditions to a nucleic acid probe comprising a six- to atleast twenty-nucleotide segment having a sequence complementary to thesix- to at least twenty-nucleotide segment of SEQ ID NO:1 or SEQ IDNO:3.

The invention even further provides isolated cDNA that hybridizes underlow-stringency conditions to a nucleic acid probe comprising a sequencecomplementary to the coding sequence of SEQ ID NO:1 or SEQ ID NO:3.

The invention even further provides a purified nucleic acid thathybridizes under stringent conditions to a nucleic acid probe comprisinga six- to at least a twenty-nucleotide segment of SEQ ID NO:1 or SEQ IDNO:3 or a segment having a complementary sequence to the six- to atleast twenty-nucleotide segment.

The invention even further provides purified nucleic acid thathybridizes under low-stringency conditions to a nucleic acid probecomprising the coding sequence or a sequence complementary to the codingsequence of SEQ ID NO:1 or SEQ ID NO:3.

The invention even further provides a substantially pure polypeptidethat binds TIA-1 in a double transformation.

In a preferred embodiment, the isolated polypeptide has an amino acidsequence that is SEQ ID NO:2 or SEQ ID NO:4.

The invention even further provides a substantially pure polypeptidethat is immunologically reactive with monoclonal antibody 2B5 producedby a hybridoma designated ATCC #HB-11721.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents an immunoblotting analysis of TIA-1 fusion proteins.Yeast strain GGY::171 was transformed with pMA424 encoding a fusionprotein between the DNA binding domain of GAL4 and TIA-1 (rp40-GAL4DNA)or the GAL4 DNA binding domain alone (GAL4DNA). Yeast cell lysates wereprepared using 2% TRITON X-100, 100 Mm NaCl, 100 mM Tris HCl, pH 8.0, 1mM EDTA. Lysates were affinity precipitated using either poly(U)-agaroseor SEPHAROSE immobilized anti-TIA-1. After separation on a 10% SDSpolyacrylamide gel, and transfer to nitrocellulose, blots were probedwith anti-TIA-1, and developed using the ECL method.

FIG. 2A is a schematic representation of the two-hybrid system used toidentify cDNAs encoding TIA-1 binding proteins.

FIG. 2B is a schematic representation of how TIABP1 and TIABP2 arebelieved to interact with the RNA binding domains and thecarboxy-terminal auxiliary domain, respectively, of TIA-1.

FIGS. 3A, 3B, 3C, 3D and 3E give the nucleotide sequence (SEQ ID NO:1)and deduced amino acid sequence (SEQ ID NO: 2) of TIABP1.

FIGS. 4A, 4B, 4C, 4D, 4E and 4F give the nucleotide sequence (SEQ IDNO:3) and deduced amino acid sequence (SEQ ID NO:4) of TIABP2.

FIG. 5 is a comparison of the deduced amino acid sequence of TIABP1 withthe amino acid sequences of known E2-type ubiquitin conjugating enzymes.Amino acids common to all ubiquitin conjugating enzymes are depicted inbold type. In FIG. 5, HHR6B means human homolog of RAD6 {Koken M. H. M.et al, (1991) "Structural and Functional Conservation of Two HumanHomologs of the Yeast DNA Repair Gene RAD6", Proc. natl. Acad. Sic.,88:8865-8869}; Human E2 means E2-Type ubiquitin conjugating enzyme;HHR6A means Human homolog of RAD6 {Koken, M. H. M. et al, supra (1991)};Dhr6 means Drosophila homolog of RAD6 {Koken M. et al, (1991), "Dhr6, aDrosophila homolog of the yeast DNA-repair gene RAD6", Proc. Natl. Acad.Sci., 88:383203836}; rhp6 means RAD6 homolog in pombe {Reynolds P. etal, (1990), "The rhp6.sup.± gene of Schizosaccharomyces pombe: AStructural and Functional Homolog of the RAD6 Gene from the DistantlyRelated Yeast Saccharomyces cerevisiae", EMBO J., 9:1423-1430}; and RAD6means radiation mutant number 6 {Jentsch S. et al, (1987), "The YeastDNA Repair Gene RAD6 Encodes a Ubiquitin-conjugating Enzyme", Nature,329:131-134}.

FIGS. 6A and 6B are schematic representations of the two-hybrid systemused to screen for drugs inhibiting the interaction between TIA-1 andTIABP1.

FIG. 7 is a Northern blotting analysis that shows the expression ofmRNAs encoding TIABP2 in various tissues. Poly(A) mRNA extracted fromthe indicated human tissues was separated on a 1% formaldehyde agarosegel prior to transferring to nitrocellulose. The blot was then probedwith a complete cDNA encoding TIABP2. The relative migrations of RNAsize markers are shown on the left.

FIGS. 8A, 8B and 8C are a comparison of the deduced amino acid sequenceof TIABP2 (TIAK) with several known protein kinases. Consensus sequencescorresponding to the 10 signature motifs that define protein kinases areindicated below the sequences. Consensus sequence V is omitted.Asterisks over the TIABP2 (TIAK) sequences indicate amino acids that areshared by TIABP2 (TIAK) and the HSV-2 kinase ICP10. Peptide insertsfound in the TIABP2 (TIAK) sequence that are absent from the srcsequence are indicated by lines labeled A through G.

FIGS. 9A, 9B, and 9C depict expression of recombinant TIABP2 in Coscells.

For FIG. 9A, Cos cells transformed with pMT2 (TIABP2) were lysed inNP-40 lysis buffer. Lysates were then immunoprecipitated with monoclonalantibodies reactive with the HA tag (anti-HA), TIABP2 (anti-2B5designated anti-TIAK) or an isotype-matched control antibody.Immunoprecipitates were then subjected to an in vitro kinase assay{Parker, R., et al (1984), "Expression of v-src and chicken c-src in ratcells demonstrates qualitative differences between pp60 v-src and pp60c-src", Cell, 37:131}, separated on a 10% SDS polyacrylamide gel, andexposed for autoradiography. A prominent 65 kD protein which is the sizeexpected of the hemagglutinin tagged TIABP2 molecule is identified inthese autoradiograms (arrow). Lower molecular weight phosphoproteinsmight be proteolytic degradation products of the full length TIABP2kinase. The relative migration of molecular-size markers is shown at theleft.

For FIG. 9B, Cos cell lysates prepared from cells transformed with pMT2(HA-TIAPB2) (here designated Cos (HA-TIAK)) or the PMT vector alone (Cos(vector)), were immunoprecipitated with monoclonal antibodies reactivewith the hemagglutinin tag (anti-HA) or with an isotype-matched controlmonoclonal antibody. Affinity precipitates were separated on a 10% SDSpolyacrylamide gel, transferred to PVDF membranes, and then subjected toa renaturation procedure followed by the addition of ³² Pγ ATP. Afterwashing the filters, they were exposed for autoradiography. Theautophosphorylated TIABP2 kinase was identified as a 65 kDphosphoprotein (arrow), confirming the intrinsic kinase activity ofTIABP2. The autophosphorylated kinase was then excised from the PVDFfilter and subjected to amino acid hydrolysis. Hydrolyzed amino acidswere then separated on a two-dimensional electrophoresis, thin-layerchromatography apparatus (FIG. 9C). The relative migration of standardsfor phosphoserine (PS), phosphothreonine (PT) and phosphotyrosine (PY)are indicated. This analysis confirms that TIABP2 is a serine/threoninekinase.

FIG. 10A, 10B, and 10C characterize natural TIABP2.

FIG. 10A is an immunoprecipitation of natural TIABP2 from lysates ofHeLa cells and K562 cells. Immunoprecipitates were prepared using amonoclonal antibody reactive with TIABP2 (anti-2B5, here designatedanti-TIAK) or with an isotype-matched control monoclonal antibody.Immunoprecipitates were subjected to the in vitro kinase assay prior toseparation on a 10% SDS polyacrylamide gel. After transferring tonitrocellulose membranes, autoradiograms revealed a phosphorylateddoublet centered around 65 kD which was specifically observed inimmunoprecipitates prepared with antibodies reactive with TIABP2. Insome cells (such as K562 shown in this figure), immunoprecipitatessubjected to the in vitro kinase assay also included additionalphosphoproteins migrating at 50 kD, 34 kD, and 21 kD. The identity ofthese candidate TIABP2 substrates is unknown. FIG. 10B shows thatnatural TIABP2 is a constitutively phosphorylated protein. In thisexperiment, Jurkat cells labeled with ³² P-orthophosphate were lysedwith NP-40 lysis buffer and immunoprecipitated with a monoclonalantibody reactive with TIABP2 (anti-2B5, here designated anti-TIAK) orwith an isotype-matched control antibody. The monoclonal antibodyreactive with TIABP2 specifically precipitated a phosphorylated doubletcentered around 65 kD (arrows). When these phosphorylated bands wereexcised from the gel and subjected to amino acid hydrolysis, naturalTIAK was found to be phosphorylated exclusively on serine and threonineresidues (FIG. 10C).

FIG. 11 shows the physical association between TIABP2 and TIA-1. Wholecell lysates prepared from Cos transformants expressing HA-TIABP2 wereseparated on a 10% SDS-polyacrylamide gel (lane 1), or affinityprecipitated using mAb 2B5 (lane 2), immobilized GST (lane 3),GST-p15-TIA-1 (lane 4), or GST-p40-TIA-1 (lane 5). After transferring tonitrocellulose, the blot was probed with anti-2B5, a mAb reactive withTIABP2. The relative migration of molecular size markers is shown at theleft.

FIG. 12 shows the effects of p15-TIA-1 on the kinase activity of TIABP2.Lysates prepared from Cos cells transformed with TIABP2 wereimmunoprecipitated using anti-2B5, and subjected to the in vitro kinaseassay in the presence of 5 μg/ml GST (lane 1), 5 μg/ml GST-fyn-SH3 (afusion protein encoding GST at the amino terminus and the SH3 bindingdomain of the fyn kinase at the carboxyl terminus), (lane 2), 5 μg/mlGST-TIAR (a fusion protein between GST and the TIA-1 related proteinTIAR), (lane 3), 1 μg/ml GST-p15-TIA-1 (lane 4), 5 μg/ml GST-p15-TIA-1(lane 5), 10 μg/ml GST-p15-TIA-1 (lane 6), or 20 μg/ml GST-p15-TIA-1(lane 7) as described in the Detailed Description of the Inventionsection. The relative migration of molecular-size markers is shown atthe left. The relative migration of phosphorylated substrates is shownat the right.

DETAILED DESCRIPTION OF THE INVENTION

A family of cytotoxic granule-associated RNA-binding proteins (TIA-1 andTIAR) that appear to be involved in lymphocyte mediated cytolysis havebeen identified and are described in U.S. Pat. Nos. 5,079,343,5,298,407, and 5,340,935 (all three of which are expressly incorporatedherein by reference). The ability of purified recombinant TIA-1 and TIARto induce DNA fragmentation in digitonin-permeabilized thymocytessuggests that these molecules activate an endogenous pathway ofprogrammed cell death in CTL targeted cells. The molecular interactionsby which TIA-1 and TIAR trigger programmed cell death are unknown. Thepresent inventors have employed a genetic approach to identify molecularsubstrates for TIA-1 that might be involved in the programmed cell deathpathway. Using the two hybrid system, the present inventors haveisolated two distinct cDNAs encoding TIA-1 binding proteins and havedesignated them TIABP1 and TIABP2. TIABP1 and TIABP2 interact with theRNA-binding domain and the carboxy-terminal auxiliary domain of TIA-1,respectively. The deduced amino acid sequence of TIABP2 is related tothe protein kinases and the deduced amino acid sequence of TIABP1reveals it to be a member of a family of E2-type ubiquitin-conjugatingenzymes. Because the ubiquitin pathway has been implicated in suchdiverse biologic processes as spermatogenesis, sporulation, DNA repairand programmed cell death, the interaction between TIA-1 and TIABP1 isexpected to directly, or indirectly, trigger the programmed cell deathpathway. Surprisingly, the present inventors found that p53, a tumorsuppressor molecule that is also involved in triggering programmed celldeath, also interacts with the ubiquitin conjugating enzyme TIABP1.Because the ubiquitin-mediated degradation of p53 is thought to beessential for malignant transformation induced by papilloma viruses,drugs that disrupt the interaction between TIABP1 and p53 are expectedto have anti-tumor activity against HPV-induced human cancers.

The present invention includes an isolated cDNA comprising a sequencethat encodes a polypeptide that binds TIA-1 in a double transformation.The isolated cDNA includes a sequence that encodes a polypeptide thatbinds the RNA binding domain and another sequence that binds thecarboxy-terminal auxiliary domain of TIA-1.

The polypeptide that binds the carboxy-terminal auxiliary domain also isimmunologically reactive with monoclonal antibody 2B5 produced by thehybridoma designated ATCC #HB-11721.

In a preferred embodiment, the isolated cDNA has a sequence, and thecDNA sequence encodes a polypeptide that is substantially identical toSEQ ID NO:1 or to SEQ ID NO:3.

Plasmids carrying the cDNA having SEQ ID NO:1 and SEQ ID NO:3 weredeposited on Jul. 30, 1993, at the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Mass. 20852 under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the purposes of Patent Procedure. The plasmids weredesignated ATCC #69371 and ATCC #69372, respectively.

A hybridoma that produces a monoclonal antibody that reacts with TIABP2and fragments of TIABP2 was deposited on Sep. 27, 1994; also at the ATCCunder the terms of the Budapest Treaty. The hybridoma was designatedATCC #HB-11721.

The term "polypeptide" as used herein means a mature protein, precursorsof the mature protein and fragments of either.

The phrase "isolated complementary DNA (cDNA)" as used herein isintended to denote a DNA molecule that is complementary to a naturallyoccurring mRNA encoding the TIA-1 binding proteins, and that has beenengineered or synthesized so that the polypeptide-encoding sequence itincludes is not flanked by the genes which, in the naturally-occurringgenome of the organism from which such polypeptide-encoding sequenceoriginated, normally flank such sequence.

The phrase "purified nucleic acid" as used herein means an RNA or DNAmolecule that is substantially free of those other nucleic acidmolecules with which it naturally associates within a cell: e.g., lessthan 30% of the purified nucleic acid preparation is made up of suchcontaminating naturally-occurring molecules. Either a purified nucleicacid or an isolated cDNA may be produced, for example, by cloning afragment of genomic DNA, by creating a cDNA from a mRNA template, or bysynthetically manufacturing a nucleic acid of the appropriate sequence.

The phrase "stringent conditions" as used herein to describehybridization means the conditions described in Sambrook et al,Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989. "Low-stringency conditions" as usedherein to describe hybridization means the following: Prehybridizationin 50% formamide, 5×SSC, 25 mM potassium phosphate buffer (pH 7.4),5×Denhart's, and 50 μg/ml denatured salmon sperm DNA for 4-12 hours at20° C.; hybridization for 12-24 hours at 20° C.; washing in 5×SSCcontaining 0.1% SDS, at 20° C.

The phrase "binds TIA-1 in a double transformation" as used herein meansthat the polypeptide encoded by the DNA binds in a doubletransformation, as described more fully in Example I herein, in whichtwo fusion proteins are expressed, each fusion protein comprising adomain necessary for expression of a marker gene. One fusion proteincomprises the polypeptide adjacent to one of the domains and the otherfusion protein comprises TIA-1 adjacent to the other of the domains.When the polypeptide binds TIA-1, the two domains cooperate to expressthe marker gene.

The phrase "immunologically reactive" as used herein means that theantibody and antigen bind to each other (i.e., form an immune complex)with sufficient specificity to permit immunoassay of the antigen orantibody under standard conditions. The phrase does not necessarilyexclude the possibility that the antibody binds other antigens: e.g.,multimers of the antigen or related proteins as described below.

The term "TIA-1" as used herein includes within its scope naturallyoccurring TIA-1 as well as recombinant embodiments such as p40-TIA-1 andisoforms of p40-TIA-1.

The isoforms of p40-TIA-1 referred to above are those disclosed in theabove mentioned U.S. Pat. Nos. 5,079,343, 5,298,407 and 5,340,935. Theisoforms include the polypeptide designated as rp40-TIA-1 and thepolypeptide designated as rp15-TIA-1, and these may have the specificamino acid sequences set forth in the referenced patents.

The term "substantially identical", when referring to a DNA sequencethat encodes a polypeptide having a recited function means a DNAsequence that may be altered to substitute one codon coding for aspecific amino acid for another codon coding for the same amino acid aswell as a DNA sequence that has one or more nucleotide substitutions,deletions and/or insertions, but the encoded polypeptide retains itsrecited function. Thus, for example, a DNA base sequence substantiallyidentical to a sequence that encodes a polypeptide that binds p40-TIA-1or isoforms thereof could have nucleotide substitutions, deletionsand/or insertions as long as the sequence encodes a polypeptide thatbinds TIA-1. Similarly, a DNA base sequence that encodes a polypeptidethat has serine/threonine kinase activity could have nucleotidesubstitutions, deletions and/or insertions as long as the encodedpolypeptide retains its serine/threonine kinase activity.

"Substantially identical" DNA sequences include allelic variants.

Appropriate substitutions, deletions and/or insertions can be made andtested by the skilled artisan. Specifically, cDNAs encoding TIABP1 orTIABP2 can be modified to specifically delete or insert one or morecodons using site-directed mutagenesis {Foss K. and W. H. McClain,(1987), Gene, 59:285-290}. By expressing these cDNAs as fusion proteinswith, for example, the GAL4 activation domain, and producing doubletransformants as described in Example I, the resulting effect on theTIA-1 binding properties of the mutant can be determined.

DNA sequences that are "substantially identical" to the sequences codingfor TIABP1 and TIABP2 and which therefore encode proteins that retainthe ability to bind TIA-1, can be prepared in the following manner. Byusing a method known as linker scanning mutagenesis, a linker sequencerecognized by both Kpn1 and Asp718 restriction enzymes (GGTACC:Kpn1 cutsbetween cytosine residues and Asp718 cuts between guanine residues) isinserted at 30 nucleotide intervals throughout the TIABP1 and the TIABP2cDNAs. Individual linker sequences can be constructed usingoligonucleotide mediated mutagenesis which is a common method in theart. The construction of these linker scanning mutants allowsconstruction of cDNAs encoding 10 amino acid deletions throughout thecoding sequence. In a similar manner, these mutants allow insertion of arandom 10 amino acid sequence at any site within the coding region. Thissequence can be produced using oligonucleotides encoding a random aminoacid sequence which are flanked by Kpn1 Asp718. ##STR1##

The sequence can be inserted into the coding sequence by cuttingupstream linkers with Kpn1 and downstream linkers with Asp718. Eachmutant can then be tested for its ability to bind specifically to theTIA-1 protein. In this way, substantially identical cDNAs that havedeletions or insertions that do not affect the ability of their encodedproteins to interact with TIA-1 protein can be identified.

Also within the present invention is isolated cDNA that hybridizes understringent conditions, as defined above, to a nucleic acid probecomprising a six-nucleotide segment (preferably at least 10 nucleotides,and more preferably at least 20 nucleotides) having a sequencecomplementary to a six- to at least twenty-nucleotide segment of SEQ IDNO:1 or SEQ ID NO:3.

The present invention also includes an isolated cDNA that hybridizesunder low-stringency conditions, as defined above, to a nucleic acidprobe comprising a sequence complementary to the coding sequence of SEQID NO:1 or SEQ ID NO:3.

In a further embodiment, the present invention includes a purifiednucleic acid (DNA or RNA) that hybridizes under stringent conditions, asdefined above, to a nucleic acid probe comprising a six-nucleotidesegment (preferably at least 10 nucleotides, more preferably at least 20nucleotides) of SEQ ID NO:1 or SEQ ID NO:3 or a segment having acomplementary sequence to the six- to at least twenty-nucleotidesegment.

The present invention further includes a purified nucleic acid (DNA orRNA) that hybridizes under low-stringency conditions, as defined above,to a nucleic acid probe comprising the coding sequence or a sequencecomplementary to the coding sequence of SEQ ID NO:1 or SEQ ID NO:3.

The substantially pure polypeptides referred to herein as binding toTIA-1 in a double is transformation and/or as being immunologicallyreactive with monoclonal antibody 2B5 produced by hybridoma ATCC#HB-11721, are naturally occurring compounds, recombinantly producedcompounds, or synthetically prepared compounds or fragments of thecompounds. The genetically-engineered forms or those syntheticallyproduced may differ from the protein defined by SEQ ID NOS:2 and 4 byone or more (but less than about 70%) of its amino acid residues (i.e.,there should be about 30% amino acid identity), so long as they retainthe recited function.

The phrase "amino acid sequence substantially identical to" means anamino acid sequence that differs from the recited sequence by one ormore (but less than 70%) of its amino acid residues and retains thefunction of the referenced amino acid sequence as determined by routineexperimentation.

I. Isolation of cDNA Clones Encoding TIA-1 Binding Proteins

The cDNA encoding p40-TIA-1 was cloned, by known methods, into themultilinker of the publicly available pMA424 vector to produce a fusionprotein consisting of the GAL4 DNA binding domain (1-147) at the aminoterminus and p40-TIA-1 at the carboxyl terminus. Transformation of yeaststrain GGY::171 with this recombinant plasmid followed by selection onSC-His plates resulted in the efficient expression of the fusion proteinas shown in FIG. 1. Because this fusion protein lacks the GAL4activation domain, it is unable to induce β-galactosidase expression,which in GGY::171 is under control of the GAL4 promoter. Theidentification of cDNAs encoding TIA-1 binding proteins can then beaccomplished by co-transforming GGY::171 cells with pMA424 (TIA-1) andclones from a cDNA library (e.g., B cell cDNA is cloned into the Xho Isite of pSE1107) expressing fusion proteins consisting of the GAL4activation domain (768-881) at the amino terminus and peptides encodedby individual cDNAs at the carboxyl terminus (FIG. 2A). Selection fordouble transformants expressing β-galactosidase (i.e., blue colonies onX-gal plates) identifies cDNAs encoding candidate TIA-1 binding proteinsthat juxtapose the GAL4 activation domain and the GAL4 DNA bindingdomain as a consequence of the TIA-1:TIA-1-binding protein interaction.By screening the double transformants in this manner, β-galactosidaseexpressing colonies can be identified. Of these, those expressing a 1.2kb insert and those expressing a 1.5 kb insert coding for the TIA-1binding proteins (TIABP1 and TIABP2) of the present invention wereisolated.

One colony induced the expression of significantly more β-galactosidasethan the others, prompting its selection for further analysis. ThepSE1107 plasmid isolated from this colony contained a 1.5 kb cDNA insertcapable of encoding an amino acid peptide, designated TIABP2, fused tothe GAL4 transactivation domain. Because truncation mutants of TIA-1possessing only the three RNA binding domains (delta 207) did not induceβ-galactosidase expression with TIABP2, it is likely that TIABP2interacts with the carboxy-terminal protein interaction domain of TIA-1(data not shown) as schematized in FIG. 2B. Interestingly, interactionof TIABP2 with TIAR {Kawakami, A. T., et al, (1992), "Identification andfunctional characterization of a TIA-1-related nucleolysin, Proc. Natl.Acad. Sci. USA, 89:8681-8685}, a TIA-1 related RNA binding proteincapable of triggering DNA fragmentation in permeabilized thymocytes,also induced β-galactosidase expression in the yeast two-hybrid system.TIABP2 did not, however, interact with another RRM-type RNA-bindingprotein, the human poly(A)-binding protein, nor did it interact withseveral control proteins including p53 and Rb, suggesting that itsinteraction with TIA-1 and TIAR is specific. The 1.5 kb insert encodingTIABP2 was used to screen a placental cDNA library by hybridization.This resulted in isolation of a 1.8 kb cDNA. Because the firstmethionine conforms to the consensus "Kozak" sequence {Kozak, M. 1984.Compilation and analysis of sequences upstream from the translationalstart site in eukaryotic mRNAs. Nucl. Acids Res. 12:857}, it is likelyto encode the initiating methionine. Northern blots probed with this 1.8kb cDNA detected a widely expressed 1.8 kb mRNA (FIG. 7).

The nucleotide sequences (SEQ ID NO:1 and SEQ ID NO:3) and deduced aminoacid sequences (SEQ ID NO:2 and SEQ ID NO:4) of TIABP1 and TIABP2 areshown in FIGS. 3A-3E, and FIG. 4A-4F, respectively.

Because truncation mutants of TIA-1 possessing only the three RNAbinding domains (delta 207) induced β-galactosidase expression withTIABP1 but not with TIABP2, TIABP1 is believed to interact with the RNAbinding domain of TIA-1. Further, the deduced amino acid sequence ofTIABP1 was found to be structurally related to a family of E2 typeubiquitin activating enzymes (FIG. 5) found in the databases GenBank-76and NBRF PIR-36.

TIABP1 and TIABP2 can be expressed in prokaryotic cells, preferably E.coli, and in eukaryotic cells by methods known in the art. Both TIABP1and TIABP2 have been cloned into the pGEX vector and expressed as fusionproteins with glutathione-S-transferase. By transformation of E. coli,strain DH5, with these recombinant expression vectors, fusion peptidesincluding TIABP1 and TIABP2 were purified. In addition, both TIABP1 andTIABP2 were cloned into the pMT2 vector and used to transform Cos cellsin a transient assay. Both proteins were expressed in these cells asdemonstrated by their reactivity with both polyclonal and monoclonalantibodies specific for each polypeptide.

II. Interaction between TIABP1 and p53

E2-type ubiquitin conjugating enzymes (UCE) transfer ubiquitin to theepsilon amino groups of lysine residues on selected substrates. Althoughthe determinants of substrate specificity for individual UCEs are poorlyunderstood, individual UCEs have the potential to ubiquitinate more thanone substrate. Because of this, TIABP1 was screened for the ability tointeract with molecular substrates that might, like TIA-1, be involvedin cell cycle progression. As shown in the Table below, the tumorsuppressor gene p53 was uniquely able to interact with TIABP1 to inducethe expression of β-galactosidase in yeast transformants. Importantly,previously identified mutant forms of p53 that lacked tumor suppressoractivity (i.e., 175, 273) induced significantly less β-galactosidaseexpression. In each case, fusion proteins were designed to exclude thetransactivation domain of p53 (aa 1-73) to avoid its influence on thetranscriptional activation of β-galactosidase.

                  TABLE                                                           ______________________________________                                        Expression of β-galactosidase                                            in Yeast Double-transformants                                                 Fusion protein A                                                              (units)         Fusion protein B                                                                           β-gal                                       ______________________________________                                        GAL4DNA:p53     GAL4TA:TIABP2                                                                              0.45                                             GAL4DNA:p40-TIA-1                                                                             GAL4TA:TIABP2                                                                              142.86                                           GAL4DNA:RB      GAL4TA:TIABP2                                                                              0.33                                             GAL4DNA:RB      GAL4TA:TIABP1                                                                              0.36                                             GAL4DNA:p53 (W) GAL4TA:TIABP1                                                                              84.79                                            GAL4DNA:p53 (273)                                                                             GAL4TA:TIABP1                                                                              60.59                                            GAL4DNA:p53 (175)                                                                             GAL4TA:TIABP1                                                                              62.50                                            GAL4DNA:p40-TIA-1                                                                             GAL4TA:TIABP1                                                                              40.21                                            ______________________________________                                    

The p53 gene product is thought to block cell cycle progression at theG1/S boundary. Its expression is therefore antiproliferative, and itsinactivation appears to be required for the malignant transformation ofa number of cell types. The ubiquitin-mediated degradation of p53 hasbeen shown to be an important post-translational event in the regulationof p53 expression. In malignant transformation induced by papillomaviruses, inactivation of p53 requires the E6 viral protein, whichenhances its ubiquitin-mediated degradation. Cervical carcinomasresulting from papilloma virus infection are characterized by their lowlevels of p53 expression. If TIABP1 is specifically involved in theubiquitin-mediated degradation of p53, this interaction may be acritical step in malignant transformation.

III. Protein Kinase Activity of TIABP2

Comparison of the amino acid sequence of TIABP2 with sequences in theEMBL protein database revealed a weak similarity with a serine/threoninekinase encoded by herpes simplex viruses (HSV) 1 and 2 (FIGS. 8A-8C).This observation led the inventors to compare the amino acid sequence ofTIABP2 with signature sequences indicative of protein kinase activity(FIGS. 8A-8C). Although TIABP2 does not encode all of the "invariant"consensus residues, its sequence is similar to that of the known kinasesin each of 10 highly conserved regions (FIGS. 8A-8C). TIABP2 isexpressed in Cos cells as a fusion protein encoding an amino terminalhemagglutinin (HA) epitope tag. Immunoprecipitates prepared usinganti-HA were subjected to the in vitro kinase assay and separated on a10% SDS polyacrylamide gel. These immunoprecipitates contain theexpected 65 kD HA-TIABP2 fusion protein (FIG. 9A, arrow) indicating thatTIABP2 possesses intrinsic protein kinase activity. The protein kinaseactivity of TIABP2 was confirmed in the renaturation kinase assay shownin FIG. 9B. In this experiment, Cos cells transformed with thehemagglutinin tagged version of TIABP2 or with the vector alone werelysed with NP-40 lysis buffer and immunoprecipitated with antibodiesreactive with the HA tag. Cos cells expressing HA-TIABP2 (heredesignated HA-TIAK) specifically included a 65 kD protein which wasphosphorylated in this renaturation kinase assay (FIG. 9B, arrow). Thisassay confirms that the 65 kD phosphoprotein possesses intrinsictyrosine kinase activity and that it is not a transphosphorylationproduct of an associated protein kinase. The amino acid specificity ofthe TIABP2 kinase was determined by analysis of hydrolytic digests ofthe autophosphorylated TIABP2 kinases shown in FIG. 9C. This analysisindicates that TIABP2 is a serine/threonine kinase.

IV. Characterization of Natural TIABP2

A monoclonal antibody reactive with recombinant TIABP2 (FIG. 10A),labeled anti-TIAK), but not an isotype-matched control antibody,precipitated a doublet centered around 65 kD from both HeLa and K562lysates that were specifically labeled in the in vitro kinase assay.Immunoprecipitates prepared from K562 cell lysates also includedadditional phosphoproteins migrating at 50 kD, 34 kD, and 21 kD.Although the identity of these associated proteins is unknown, they arepossible substrates for the kinase activity of TIABP2. Natural TIABP2expressed in Jurkat cells was found to be a constitutivelyphosphorylated protein which migrated as a doublet centered around 65 kD(FIG. 10B, arrows). The constitutive phosphorylation of TIABP2 occurredexclusively on serine and threonine residues as shown in thephosphoamino acid analysis shown in FIG. 10C.

V. Physical Interaction between TIA-1 and TIABP2

Results obtained using the yeast two-hybrid system suggested a specificinteraction between TIABP2 and the protein interaction domain of TIA-1.These results were confirmed by showing that TIABP2 contained in lysatesfrom Cos transformants could be specifically co-precipitated by GSTfusion proteins expressing the protein interaction domain of TIA-1. FIG.11 shows that lysates prepared from Cos cells transformed with TIABP2contain a 65 kD protein that is recognized by a monoclonal antibodyreactive with TIABP2 (lane 1). Affinity precipitates prepared usingglutathione beads coupled to GST did not contain the 65 kD recombinantTIABP2 protein (lane 2). Affinity precipitates prepared usingglutathione beads coupled to either GST-p15-TIA-1 (lane 3) orGST-p40-TIA-1 (lane 4) included the 65 kD TIABP2 protein. This result isconsistent with results obtained using the two hybrid system, andsuggest that TIABP2 interacts with the carboxy terminal proteininteraction domain of TIA-1.

VI. Regulation of TIABP2 by TIA-1

Cos cells transformed with a cDNA encoding TIABP2 were lysed with NP-40lysis buffer and immunoprecipitated using anti-2B5. Theseimmunoprecipitates were subjected to the in vitro kinase assay in thepresence of GST-fusion proteins encoding either control peptides orp15-TIA-1 (FIG. 12). Each of these immunoprecipitates expressed a 65 kDphosphoprotein migrating in the position expected for TIABP2 (in FIG. 12designated TIAK). In the presence of GST alone or a GST-fusion proteinencoding the SH3 domain of the fyn tyrosine kinase, additionaltransphorylated substrates were not identified. However, in the presenceof GST-fusion proteins encoding p15-TIA-1, the appearance oftransphosphorylated substrates migrating at 34 kD and 21 kD were inducedin a dose-dependent manner. The 21 kD phosphoprotein was not observed atthe highest concentration of GST-p15-TIA-1 (20 μg/ml). At thisconcentration, the GST-p15-TIA-1 itself became a target forphosphorylation, suggesting that competition for phosphorylation of thetwo proteins might be responsible for this result. Theautophosphorylation of TIABP2 was not changed in the presence or absenceof GST-p15-TIA-1. These results suggest that TIA-1 can alter the abilityof TIABP2 to transphosphorylate associated substrates.

VII. Use

The ability of TIA-1 to enhance the protein kinase activity of TIABP2suggests that the activation of TIABP2 may be required for the inductionof apoptotic cell death. Because the kinase activity of TIABP2 can beeasily measured in vitro, it will be possible to screen for small drugswhich either activate or inhibit the activity of this serine/threoninekinase. It will also be possible to screen for small drugs that disruptthe specific association between TIA-1 and TIABP2 that is likely tooccur during CTL-mediated killing of target cells. Such drugs would beexpected to have protective activity against inflammatory conditions inwhich TIA-1-mediated killing of target cells induced by cytotoxic Tlymphocytes is important in the pathophysiology of disease. Examples ofsuch diseases would include graft vs. host disease of the skin, renalallograft rejection, and all transplantation organ rejections.

TIA-1 is a cytotoxic granule-associated RNA binding protein that is acandidate toxin used by cytotoxic lymphocytes in the destruction oftarget cells. Although the molecular mechanisms responsible for thetoxic effects of TIA-1 are unknown, the ability of purified recombinantTIA-1 to induce DNA fragmentation in permeabilized target cells suggeststhat this protein might induce apoptotic death in cells into which it isintroduced. Target cell proteins that interact with TIA-1 are candidatesubstrates in a molecular cascade leading to target cell death. As such,cDNAs encoding TIABP1 and TIABP2 and the recombinant proteins that theyencode, can be used in in vitro assays to search for drugs with theability to disrupt the specific interaction between TIA-1 and theindividual TIABPs. One example of such an application is outlined inExample II.

Because TIABP1 is an E2-type ubiquitin conjugating enzyme, it is likelyto be involved in the ubiquitin-mediated degradation of TIA-1. Theexpression of toxic molecules such as TIA-1 must be closely regulated inthe cell to prevent unwanted toxic effects. The present inventors haveobserved TIA-1 to be rapidly degraded in an ubiquitin-dependent mannerin rabbit reticulocyte lysates. If a TIABP1 homolog is specificallyinvolved in this process, then the regulation of the TIABP1 proteinitself might be important in regulating the expression of TIA-1 as well.Using cDNAs reactive with TIAPB1 and the polyclonal antisera reactivewith the recombinant and natural TIAPB1 protein, it will be possible toscreen for transcriptional regulators that turn off the expression ofthis regulatory protein. Such a compound might be expected to result inincreased expression of TIA-1 and, consequently, death of the cell. Ifcompounds can be isolated which are preferentially taken up by rapidlygrowing cells, then such compounds could be used as anti-cancer agents.Because TIAPB1 may also regulate the expression of the tumor suppressorgene p53, its decreased expression might also result in an increase inp53 protein, thus triggering apoptosis in a rapidly growing cell.

Purified recombinant TIABP2 protein and cDNAs encoding TIABP2 willallow, in an analogous fashion, screening for small drugs that caneither disrupt or enhance the specific association between TIA-1 andTIABP2. Given the potential role of TIA-1 as a molecular toxin, suchagents would be candidate anti-cancer drugs.

EXAMPLES

The present invention will now be described by reference to specificexamples which are not meant to limit the invention in any way.

Example I ISOLATION AND CHARACTERIZATION OF TWO cDNA CLONES ENCODINGTIA-1 BINDING PROTEINS TIAPB1 AND TIABP2

The yeast expression vector PVA424 was digested with the restrictionenzymes EcoR1 and BamH1 which cut within the multilinker regionfollowing the GAL4 DNA binding domain. The linearized vector wasisolated by electrophoresis in a 1% low-melt agarose gel, after whichthe band was visualized by transillumination and excised from the gel.The cDNA encoding p40-TIA-1 was excised from the pSP65(λ269.4) vector bya double digestion with BstEII and BamHI. After electrophoreticseparation on a 1% low-melt agarose gel, the smaller piece of linear DNAwas excised from the gel. The two excised DNA fragments were combinedwith a synthetic oligolinker of the sequence:

(EcoRI) AAGTCGTCG (SEQ ID NO:12)

GCAGCCATTG (BstEII) (SEQ ID NO:13)

encoding an EcoR1 site at the upstream and a BstEII site at thedownstream end. Following ligation, the full-length plasmid designatedPMA424(p40-TIA-1) was isolated, expanded and purified.

Transformation of yeast strain GGY::171 with this recombinant plasmidwas achieved by the lithium acetate method as described in Nucleic AcidResearch, (1991), 19:5791. Following selection on SC-his dropout plates,individual transformants were analyzed for their expression of thep40-TIA-1-Gal4 DNA binding domain fusion protein as shown in FIG. 1.Lysates from yeast transformed with the recombinant p40-GAL4 DNA bindingdomain contained a protein migrating at approximately 65 kD which wasrecognized by both poly(U)-agarose and by a monoclonal antibody reactivewith TIA-1. Conversely, lysates from yeast cells transformed withvectors encoding the GAL4 DNA binding domain alone did not contain thisimmunoreactive material. In both cases, yeast cell lysates were preparedusing 2% TRITON X-100, 100 mM NaCl, 100 mM Tris HCl pH 8.0, 1 mM EDTA.Following affinity precipitation using either poly(U)-agarose orSEPHAROSE immobilized anti-TIA-1 antibodies, precipitates were separatedon a 10% SDS polyacrylamide gel and transferred to nitrocellulose.Individual blots were then probed with the monoclonal antibody reactivewith TIA-1 and developed using the ECL method.

The identification of cDNAs encoding TIA-1 binding proteins was thenaccomplished by co-transforming GGY::171 cells with PMA424(p40-TIA-1)and a cDNA library prepared from poly(A) RNA from human B cells fromwhich cDNA was transcribed and cloned into the XhoI site of the pSE1107vector. In the cDNA library, individual cDNAs were expressed as fusionproteins consisting of the GAL4 activation domain (residues 768-881) atthe amino terminus and peptides encoded by individual cDNAs at thecarboxyl terminus as diagrammed in FIG. 2A. Following cotransfection,cells were plated on SC-Leu-His dropout medium plates to select fordouble transformants. After three days at 30° Celsius, yeast colonieswere replica plated to Sc-Leu-His dropout medium plates containing X-galfor selection of colonies expressing β-galactosidase. Positive colonieswere selected and expanded. To isolate DNA from positive colonies,individual colonies were suspended in 100 μl of lysis buffer (2% TRITONX-100, 1% SDS, 100 mM NaCl, 100 mM Tris HCl pH 8.0, 1 mM EDTA), plus 100μl of phenol/chloroform/isoamyl alcohol. After the addition of 0.1 gramof glass beads, these preparations were vortexed for two minutes,centrifuged in an Eppendorf centrifuge, and the supernatants weretransferred to a clean Eppendorf tube. DNA was then precipitated by theaddition of 3M NaOAc, 250 μl ethanol. After resuspending theprecipitated DNA in 4 μl TE buffer, 2 μl of this DNA was used totransform an E. coli LeuB⁻ strain (W921) by electroporation. DNA wasisolated from E. coli transformants and digested with XhoI to liberatethe cDNA inserts. After screening 400,000 double transformants of yeastcells, four positive colonies were obtained. Three of these encodedTIAPB1 (1.2 kb insert), and one encoded TIABP2 (1.5 kb insert).

The nucleotide sequence (SEQ ID NO:1) and deduced amino acid sequence(SEQ ID NO:2) of TIABP1 are shown in FIGS. 3A, 3B, 3C, 3D ard 3E. Thenucleotide sequence (SEQ ID NO:3) and deduced amino acid sequence (SEQID NO:4) of TIABP2 are shown in FIG. 4A-4F.

GST Fusion Proteins

The 1.8 kb cDNA encoding TIABP2 was cloned into the EcoR1 site of thepolylinker region of pGEX-3× using oligonucleotide linkers. Theseconstructs were designed to express TIABP2 as a fusion protein withglutathione-S-transferase. An individual colony of E. coli (DH5)bacterial cells transformed with pGEX-3×/TIABP2 was used to inoculate 25ml of LB media containing ampicillin (100 μg/ml). Cultures were grownwith shaking at 37° C. overnight. 20 ml of the overnight culture wasused to inoculate 800 ml of 2×YT medium containing 100 μg/ml ampicillin.Cultures were shaken at 37° C. until the O.D.₆₀₀ was approximately 0.6.At that time, IPTG was added to 0.2 mM final concentration and cultureswere incubated for a further 3 hours at 30° C. Cells were then harvestedby centrifugation at 4,000 rpm for 10 min. Pellets were suspended in 10ml of PBS containing 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF. Cells were thendisrupted by sonication, and centrifuged at 40,000 rpm for 30 min at 4°C. to remove insoluble debris. Supernatants were applied to a column ofglutathione-agarose beads (Sigma Chemical Company) and incubated for 30min at 4° C. Beads were then washed 3× with 10 ml of PBS containing 1 mMEDTA, 1 mM DTT, 0.1 mM PMSF, and 2× with PBS alone. Individual fusionproteins were then eluted by competition with glutathione applied at 10mM final concentration in 50 mM Tris, pH 8.0. The eluate was dialyzedagainst 50 mM Tris, 150 mM NaCl, 1 mM DTT, pH 8.0, to remove freeglutathione. The purified fusion protein was analyzed on a 10% SDSpolyacrylamide gel by staining with Coomassie blue. Fusion proteins werealso analyzed by immunoblotting using rabbit polyclonal antisera raisedagainst recombinant TIABP2.

Northern Blot Analysis

Nitrocellulose filters containing poly(A)+RNA from the indicated tissueswere purchased from Clontech. Each filter was prehybridized in 50%formamide, 5×SSC, 25 mM potassium phosphate buffer (pH 7.4), 5×Denhart'sand 50 mg/ml denatured salmon sperm DNA for 4 hours at 42° C. The 1.8 kbTIABP2 insert DNA was ³² P-labeled by nick translation, diluted in theabove solution, and hybridized to the filter for 24 hours at 42° C. Thefilter was then washed twice with 1×SSC containing 0.1% SDS and twicewith 0.1×SSC containing 0.1% SDS prior to autoradiographic exposure.

Cos Cell Transfections

Cos cells were transfected with the plasmid pMT-2 containing theindicated insert DNA using the diethylaminoethyl dextran method asdescribed by Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989.Molecular Cloning. A Laboratory Manual. After three days of culture,transfected cells were solubilized with lysis buffer, and used in theimmunoprecipitation and immunoblotting experiments.

Immunoprecipitations

The indicated cell types were lysed in NP-40 lysis buffer (1% NP-40, 150mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 50 mM Tris HCl,pH 8.0), and immunoprecipitations were performed using methodspreviously described: Anderson, P., et al (1990), "A monoclonal antibodyreactive with a 15 kD cytoplasmic granule-associated protein defines asubpopulation of CD8+ T-lymphocytes", Journal of Immunology, 144:574.Individual immunoprecipitates were separated on a 10% SDS polyacrylamidegel, transferred to nitrocellulose or PVDF filters and revealed anddeveloped using polyclonal and monoclonal antibodies as described below.

Immunoblot Analysis

Immunoblotting analysis was carried out as previously described{Anderson, P. et al, (1990), "A monoclonal antibody reactive with a15-kDa cytoplasmid granule-associated protein defines a subpopulation ofCD8+ T lymphocytes", J. Immunol. 144:574}. Immunoblots were developedusing polyclonal or monoclonal antibodies reactive with TIABP2, followedby horse radish peroxidase conjugate protein A/G. Blots were revealedusing the ECL detection system (Renaissance, DuPont, Boston, Mass.).

Example II METHOD TO SCREEN FOR INHIBITORS OF TIABP1:P53 OR TIA-1INTERACTIONS

Existing technology can be used to screen for drugs that inhibit theinteraction between TIAPB1 and its substrates. In the case of p53:TIABP1interactions, such drugs might have anti-tumor activity directed againstHPV-associated cancers expressing low levels of p53. In the case ofTIA-1:TIAPB1 interactions, such drugs might be beneficial in treatingautoimmune diseases in which CTLs are involved in tissue destruction.Examples include graft vs. host disease, allograft rejection followingorgan transplantation, autoimmune thyroiditis, and autoimmune diabetesmelitis.

A variation of the two hybrid system adapted for mammalian cells toscreen for inhibitors of TIABP1:TIA-1, TIAPB1:p53, and TIABP2:TIA-1interactions can be employed. The method to be employed is schematizedin FIGS. 6A and 6B. The general method involves the construction ofplasmids encoding chimeric fusion proteins whose interaction triggersthe transcription of a reporter gene in a mammalian cell. One of severalpromoters, reporter genes, DNA binding proteins, and transactivationdomains can be used. In one example (FIG. 6A), plasmids encodingchimeric fusion proteins between: i) the GAL4 DNA-binding domain andTIA-1, and ii) TIABP1 and the VP16 activation domain (411-455) are usedto activate transcription of the gene for secreted alkaline phosphataseunder control of the GAL4 promoter. In this example, the interactionbetween TIA-1 and TIAPB1 results in constitutive expression of secretedalkaline phosphatase. By culturing these cells in the presence ofcandidate inhibitors of the TIA-1:TIAPB1 interaction, cell supernatantscan be screened for decreased alkaline phosphatase activity. In anotherexample (FIG. 6B), plasmids encoding fusion proteins between: i) thetetracycline repressor and TIA-1, and ii) TIAPB1 and the V16transactivation domain (411-455) are used to transform cells expressinga toxin gene such as ricin A under control of a tetracycline promoter.Cells are then cultured in the presence of tetracycline, which preventsthe interaction between the tetR and the tet promoter. At confluence,tetracycline is removed and individual drugs are added. The interactionbetween TIA-1 and TIAPB1 results in transcription of ricin A, resultingin cell death. Cells cultured in the presence of drugs which block theinteraction between TIA-1 and TIAPB1 will survive. A vital dye can beused to screen for viable cells.

Example III PURIFICATION AND ISOLATION OF TIAPB1 AND TIABP2

cDNAs encoding TIABP1 and TIABP2 were cloned into one EcoR1 site of thepolylinker region of pGEX-3× using oligonucleotide linkers. Theseconstructs were designed to express both TIAPB1 and TIABP2 as fusionproteins with glutathione-S-transferase. Each recombinant plasmid wastransfected into E. coli (DH5) and fusion proteins s were induced by theaddition of IPTG. Individual colonies of DH5 bacterial cells transformedwith either pGEX-3×/TIAPB1 or pGEX-3×/TIABP2 were used to inoculate 25ml of LB media containing ampicillin (100 μg/ml). Cultures were grownwith shaking at 37° C. overnight. 20 ml of the overnight culture wasused to inoculate 800 ml of 2×YT medium containing 100 μg/ml ampicillin.Cultures were shaken at 37° C. until the O.D.₆₀₀ was approximately 0.6.At that time, IPTG was added to 0.2 mM final concentration and cultureswere incubated for a further 3 hours at 30° C. Cells were then harvestedby centrifugation at 4,000 rpm for 10 min Pellets were suspended in 10ml of PBS containing 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF. Cells were thendisrupted by sonication, and centrifuged at 40,000 rpm for 30 min at 4°C. to remove insoluble debris. Supernatants were applied to a column ofglutathione-agarose beads (Sigma Chemical Company) and incubated for 30min at 4° C. Beads were then washed 3× with 10 ml of PBS containing 1 mMEDTA, 1 mM DTT, 0.1 mM PMSF, and 2× with PBS alone. Individual fusionproteins were then eluted by competition with glutathione applied at 10mM final concentration in 50 mM Tris, pH 8.0. The eluate was dialyzedagainst 50 mM Tris, 150 mM NaCl, 1 mM DTT, pH 8.0, to remove freeglutathione. The purified fusion protein was analyzed on a 10% SDSpolyacrylamide gel by staining with Coomassie blue. Fusion proteins werealso analyzed by immunoblotting using rabbit polyclonal antisera raisedagainst recombinant TIA-1 and recombinant TIAR.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 21                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1206 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 172..648                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCACCAAACCCAAAAAAAGAGATCTGGAATTCGGATCCTCGAGGCCACGAAGGCCGCGGG60                CTCCGGAGGGAAGTCCCGAGACAAAGGGAAGCGCCGCCGCCGCCGCCCCGCTCGGTCCTC120               CACCTGTCCGCTACGCTCGCCGGGGCTGCGGCCGCCCGAGGGACTTTGAACATGTCG177                  MetSer                                                                        GGGATCGCCCTCAGCAGACTCGCCCAGGAGAGGAAAGCATGGAGGAAA225                           GlyIleAlaLeuSerArgLeuAlaGlnGluArgLysAlaTrpArgLys                              51015                                                                         GACCACCCATTTGGTTTCGTGGCTGTCCCAACAAAAAATCCCGATGGC273                           AspHisProPheGlyPheValAlaValProThrLysAsnProAspGly                              202530                                                                        ACGATGAACCTCATGAACTGGGAGTGCGCCATTCCAGGAAAGAAAGGG321                           ThrMetAsnLeuMetAsnTrpGluCysAlaIleProGlyLysLysGly                              35404550                                                                      ACTCCGTGGGAAGGAGGCTTGTTTAAACTACGGATGCTTTTCAAAGAT369                           ThrProTrpGluGlyGlyLeuPheLysLeuArgMetLeuPheLysAsp                              556065                                                                        GATTATCCATCTTCGCCACCAAAATGTAAATTCGAACCACCATTATTT417                           AspTyrProSerSerProProLysCysLysPheGluProProLeuPhe                              707580                                                                        CACCCGAATGTGTACCCTTCGGGGACAGTGTGCCTGTCCATCTTAGAG465                           HisProAsnValTyrProSerGlyThrValCysLeuSerIleLeuGlu                              859095                                                                        GAGGACAAGGACTGGAGGCCAGCCATCACAATCAAACAGATCCTATTA513                           GluAspLysAspTrpArgProAlaIleThrIleLysGlnIleLeuLeu                              100105110                                                                     GGAATACAGGAACTTCTAAATGAACCAAATATCCAAGACCCAGCTCAA561                           GlyIleGlnGluLeuLeuAsnGluProAsnIleGlnAspProAlaGln                              115120125130                                                                  GCAGAGGCCTACACGATTTACTGCCAAAACAGAGTGGAGTACGAGAAA609                           AlaGluAlaTyrThrIleTyrCysGlnAsnArgValGluTyrGluLys                              135140145                                                                     AGGGTCCGAGCACAAGCCAAGAAGTTTGCGCCCTCATAAGCAGCGA655                             ArgValArgAlaGlnAlaLysLysPheAlaProSer                                          150155                                                                        CCTTGTGGCATCGTCAGAAGGAAGGGATTGGTTTGGCAAGAACTTGTTTACAACATAATC715               TAAAGTTGCTCCATACATGACTAGTCACCTGGGGGGGTTGGGCGGGCGCATCTTCCATTG775               CCGCCGCGGGTGTGCGTCTCGATTCGCTGAATTGCCCGTTTCCATACAGGGTCTCTTCCT835               TCGGTCTTTTGTATTTTTGATTGTTATGTAAAACTCGCTTTTATTTTAATATTGATGTCA895               GTATTTCAACTGCTGTAAAATTATAAACTTTTATACTTGGGTAAGTCCCCAGGCGAGGTT955               CCTCGCTCTGGGATGCAGGCATGCTTCTCACGTGCAGCTGTCAACTTGGCCTCAGCTGGC1015              TGTATGGAAATGCACCCTCCCTCCTGCGCTCCTCTCTAGAACCGGCTAGAACCTGGGCTG1075              TGCTGCTTTTGAGCCTCAGACCCCAGGGCAGCATCTCGGTTCTGCGCCACTTCCTTTGTG1135              TTTATATGGCGTTTTGTCTGTGTTGCTGTTTAGAGTAAATAAAACTGTTTATATAAAAAA1195              AAAAAAAAAAA1206                                                               (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 158 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetSerGlyIleAlaLeuSerArgLeuAlaGlnGluArgLysAlaTrp                              151015                                                                        ArgLysAspHisProPheGlyPheValAlaValProThrLysAsnPro                              202530                                                                        AspGlyThrMetAsnLeuMetAsnTrpGluCysAlaIleProGlyLys                              354045                                                                        LysGlyThrProTrpGluGlyGlyLeuPheLysLeuArgMetLeuPhe                              505560                                                                        LysAspAspTyrProSerSerProProLysCysLysPheGluProPro                              65707580                                                                      LeuPheHisProAsnValTyrProSerGlyThrValCysLeuSerIle                              859095                                                                        LeuGluGluAspLysAspTrpArgProAlaIleThrIleLysGlnIle                              100105110                                                                     LeuLeuGlyIleGlnGluLeuLeuAsnGluProAsnIleGlnAspPro                              115120125                                                                     AlaGlnAlaGluAlaTyrThrIleTyrCysGlnAsnArgValGluTyr                              130135140                                                                     GluLysArgValArgAlaGlnAlaLysLysPheAlaProSer                                    145150155                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1776 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 19..1668                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GGCGGACTCGGTGGCTAGCCGATGAGGAGGCCGCGGGGGGAACCCGGC48                            ProMetArgArgProArgGlyGluProGly                                                1510                                                                          CCCCGGGCCCCGAGACCGACTGAGGGAGCGACCTGCGCAGGGCCCGGG96                            ProArgAlaProArgProThrGluGlyAlaThrCysAlaGlyProGly                              152025                                                                        GAGTCATGGTCTCCATCACCCAACTCCATGCTTCGAGTCCTGCTCTCT144                           GluSerTrpSerProSerProAsnSerMetLeuArgValLeuLeuSer                              303540                                                                        GCTCAGACCTCCCCTGCTCGGCTGTCTGGCCTGCTGCTGATCCCTCCA192                           AlaGlnThrSerProAlaArgLeuSerGlyLeuLeuLeuIleProPro                              455055                                                                        GTACAGCCCTGCTGTTTGGGGCCCAGCAAATGGGGGGACCGGCCTGTT240                           ValGlnProCysCysLeuGlyProSerLysTrpGlyAspArgProVal                              606570                                                                        GGAGGAGGCCCCAGTGCAGGTCCTGTGCAAGGACTGCAGCGGCTTCTG288                           GlyGlyGlyProSerAlaGlyProValGlnGlyLeuGlnArgLeuLeu                              75808590                                                                      GAACAGGCGAAGAGCCCTGGGGAGCTGCTGCGCTGGCTGGGCCAGAAC336                           GluGlnAlaLysSerProGlyGluLeuLeuArgTrpLeuGlyGlnAsn                              95100105                                                                      CCCAGCAAGGTGCGCGCCCACCACTACTCGGTGGCGCTTCGTCGTCTG384                           ProSerLysValArgAlaHisHisTyrSerValAlaLeuArgArgLeu                              110115120                                                                     GGCCAGCTCTTGGGGTCTCGGCCACGGCCCCCTCCTGTGGAGCAGGTC432                           GlyGlnLeuLeuGlySerArgProArgProProProValGluGlnVal                              125130135                                                                     ACACTGCAGGACTTGAGTCAGCTCATCATCCGAAACTGCCCCTCCTTT480                           ThrLeuGlnAspLeuSerGlnLeuIleIleArgAsnCysProSerPhe                              140145150                                                                     GACATTCACACCATCCACGTGTGTCTGCACCTTGCAGTCTTACTTGGC528                           AspIleHisThrIleHisValCysLeuHisLeuAlaValLeuLeuGly                              155160165170                                                                  TTTCCATCTGATGGTCCCCTGGTGTGTGCCCTGGAACAGGAGCGAAGG576                           PheProSerAspGlyProLeuValCysAlaLeuGluGlnGluArgArg                              175180185                                                                     CTCCGCCTCCCTCCGAAGCCACCTCCCCCTTTGCAGCCCCTTCTCCGA624                           LeuArgLeuProProLysProProProProLeuGlnProLeuLeuArg                              190195200                                                                     GGTGGGCAAGGGTTGGAAGCTGCTCTAAGCTGCCCCCGTTTTCTGCGG672                           GlyGlyGlnGlyLeuGluAlaAlaLeuSerCysProArgPheLeuArg                              205210215                                                                     TATCCACGGCAGCATCTGATCAGCAGCCTGGCAGAGGCAAGGCCAGAG720                           TyrProArgGlnHisLeuIleSerSerLeuAlaGluAlaArgProGlu                              220225230                                                                     GAACTGACTCCCCACGTGATGGTGCTCCTGGCCCAGCACCTGGCCCGG768                           GluLeuThrProHisValMetValLeuLeuAlaGlnHisLeuAlaArg                              235240245250                                                                  CACCGGTTGCGGGAGCCCCAGCTTCTGGAAGCCATTGCCCACTTCCTG816                           HisArgLeuArgGluProGlnLeuLeuGluAlaIleAlaHisPheLeu                              255260265                                                                     GTGGTTCAGGAAACGCAACTCAGCAGCAAGGTGGTACAGAAGTTGGTC864                           ValValGlnGluThrGlnLeuSerSerLysValValGlnLysLeuVal                              270275280                                                                     CTGCCCTTTGGGCGACTGAACTACCTGCCCCTGGAACAGCAGTTTATG912                           LeuProPheGlyArgLeuAsnTyrLeuProLeuGluGlnGlnPheMet                              285290295                                                                     CCCTGCCTTGAGAGGATCCTGGCTCGGGAAGCAGGGGTGGCACCCCTG960                           ProCysLeuGluArgIleLeuAlaArgGluAlaGlyValAlaProLeu                              300305310                                                                     GCTACAGTCAACATCTTGATGTCACTGTGCCAACTGCGGTGCCTGCCC1008                          AlaThrValAsnIleLeuMetSerLeuCysGlnLeuArgCysLeuPro                              315320325330                                                                  TTCAGAGCCCTGCACTTTGTTTTTTCCCCTGGCTTCATCAACTACATC1056                          PheArgAlaLeuHisPheValPheSerProGlyPheIleAsnTyrIle                              335340345                                                                     AGTGGCACCCCTCATGCTCTGATTGTGCGTCGCTACCTCTCCCTGCTG1104                          SerGlyThrProHisAlaLeuIleValArgArgTyrLeuSerLeuLeu                              350355360                                                                     GACACGGCCGTGGAGCTGGAGCTCCCAGGATACCGGGGTCCCCGCCTT1152                          AspThrAlaValGluLeuGluLeuProGlyTyrArgGlyProArgLeu                              365370375                                                                     CCCCGAAGGCAGCAAGTGCCCATCTTTCCCCAGCCTCTCATCACCGAC1200                          ProArgArgGlnGlnValProIlePheProGlnProLeuIleThrAsp                              380385390                                                                     CGTGCCCGCTGCAAGTACAGTCACAAGGACATAGTAGCTGAGGGGTTG1248                          ArgAlaArgCysLysTyrSerHisLysAspIleValAlaGluGlyLeu                              395400405410                                                                  CGCCAGCTGCTGGGGGAGGAGAAATACCGCCAGGACCTGACTGTGCCT1296                          ArgGlnLeuLeuGlyGluGluLysTyrArgGlnAspLeuThrValPro                              415420425                                                                     CCAGGCTACTGCACAGACTTCCTGCTGTGCGCCAGCAGCTCTGGTGCT1344                          ProGlyTyrCysThrAspPheLeuLeuCysAlaSerSerSerGlyAla                              430435440                                                                     GTGCTTCCCGTGAGGACCCAGGACCCCTTCCTGCCATACCCACCAAGG1392                          ValLeuProValArgThrGlnAspProPheLeuProTyrProProArg                              445450455                                                                     TCCTGCCCACAGGGCCAGGCTGCCTCTAGCGCCACTACTCGAGACCCT1440                          SerCysProGlnGlyGlnAlaAlaSerSerAlaThrThrArgAspPro                              460465470                                                                     GCCCAGAGGGTGGTGCTGGTGTTGCGGGAACGCTGGCATTTCTGCCGG1488                          AlaGlnArgValValLeuValLeuArgGluArgTrpHisPheCysArg                              475480485490                                                                  GACGGCCGGGTGCTGCTGGGCTCGAGGGCCCTGAGGGAGCGGCACCTA1536                          AspGlyArgValLeuLeuGlySerArgAlaLeuArgGluArgHisLeu                              495500505                                                                     GGCCTGATGGGCTACCAGCTCCTGCCGCTACCCTTCGAGGAACTGGAG1584                          GlyLeuMetGlyTyrGlnLeuLeuProLeuProPheGluGluLeuGlu                              510515520                                                                     TCCCAGAGAGGCCTGCCCCAGCTCAAGAGCTACCTGAGGCAGAAGCTC1632                          SerGlnArgGlyLeuProGlnLeuLysSerTyrLeuArgGlnLysLeu                              525530535                                                                     CAAGCCCTGGGCCTGCGCTGGGGGCCTGAAGGGGGCTGAGGGGATGAT1680                          GlnAlaLeuGlyLeuArgTrpGlyProGluGlyGly                                          540545550                                                                     GTGGGGTTCAGGATGGCCCCCCCATGGGGGGTGGATGATTTGCACTTT1728                          GGTTCCCTGTGTTTTGATTTCTCATTAAAGTTCCTGGCCTTCAAAAAA1776                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 550 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ProMetArgArgProArgGlyGluProGlyProArgAlaProArgPro                              151015                                                                        ThrGluGlyAlaThrCysAlaGlyProGlyGluSerTrpSerProSer                              202530                                                                        ProAsnSerMetLeuArgValLeuLeuSerAlaGlnThrSerProAla                              354045                                                                        ArgLeuSerGlyLeuLeuLeuIleProProValGlnProCysCysLeu                              505560                                                                        GlyProSerLysTrpGlyAspArgProValGlyGlyGlyProSerAla                              65707580                                                                      GlyProValGlnGlyLeuGlnArgLeuLeuGluGlnAlaLysSerPro                              859095                                                                        GlyGluLeuLeuArgTrpLeuGlyGlnAsnProSerLysValArgAla                              100105110                                                                     HisHisTyrSerValAlaLeuArgArgLeuGlyGlnLeuLeuGlySer                              115120125                                                                     ArgProArgProProProValGluGlnValThrLeuGlnAspLeuSer                              130135140                                                                     GlnLeuIleIleArgAsnCysProSerPheAspIleHisThrIleHis                              145150155160                                                                  ValCysLeuHisLeuAlaValLeuLeuGlyPheProSerAspGlyPro                              165170175                                                                     LeuValCysAlaLeuGluGlnGluArgArgLeuArgLeuProProLys                              180185190                                                                     ProProProProLeuGlnProLeuLeuArgGlyGlyGlnGlyLeuGlu                              195200205                                                                     AlaAlaLeuSerCysProArgPheLeuArgTyrProArgGlnHisLeu                              210215220                                                                     IleSerSerLeuAlaGluAlaArgProGluGluLeuThrProHisVal                              225230235240                                                                  MetValLeuLeuAlaGlnHisLeuAlaArgHisArgLeuArgGluPro                              245250255                                                                     GlnLeuLeuGluAlaIleAlaHisPheLeuValValGlnGluThrGln                              260265270                                                                     LeuSerSerLysValValGlnLysLeuValLeuProPheGlyArgLeu                              275280285                                                                     AsnTyrLeuProLeuGluGlnGlnPheMetProCysLeuGluArgIle                              290295300                                                                     LeuAlaArgGluAlaGlyValAlaProLeuAlaThrValAsnIleLeu                              305310315320                                                                  MetSerLeuCysGlnLeuArgCysLeuProPheArgAlaLeuHisPhe                              325330335                                                                     ValPheSerProGlyPheIleAsnTyrIleSerGlyThrProHisAla                              340345350                                                                     LeuIleValArgArgTyrLeuSerLeuLeuAspThrAlaValGluLeu                              355360365                                                                     GluLeuProGlyTyrArgGlyProArgLeuProArgArgGlnGlnVal                              370375380                                                                     ProIlePheProGlnProLeuIleThrAspArgAlaArgCysLysTyr                              385390395400                                                                  SerHisLysAspIleValAlaGluGlyLeuArgGlnLeuLeuGlyGlu                              405410415                                                                     GluLysTyrArgGlnAspLeuThrValProProGlyTyrCysThrAsp                              420425430                                                                     PheLeuLeuCysAlaSerSerSerGlyAlaValLeuProValArgThr                              435440445                                                                     GlnAspProPheLeuProTyrProProArgSerCysProGlnGlyGln                              450455460                                                                     AlaAlaSerSerAlaThrThrArgAspProAlaGlnArgValValLeu                              465470475480                                                                  ValLeuArgGluArgTrpHisPheCysArgAspGlyArgValLeuLeu                              485490495                                                                     GlySerArgAlaLeuArgGluArgHisLeuGlyLeuMetGlyTyrGln                              500505510                                                                     LeuLeuProLeuProPheGluGluLeuGluSerGlnArgGlyLeuPro                              515520525                                                                     GlnLeuLysSerTyrLeuArgGlnLysLeuGlnAlaLeuGlyLeuArg                              530535540                                                                     TrpGlyProGluGlyGly                                                            545550                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 158 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetSerGlyIleAlaLeuSerArgLeuAlaGlnGluArgLysAlaTrp                              151015                                                                        ArgLysAspHisProPheGlyPheValAlaValProThrLysAsnPro                              202530                                                                        AspGlyThrMetAsnLeuMetAsnTrpGluCysAlaIleProGlyLys                              354045                                                                        LysGlyThrProTrpGluGlyGlyLeuPheLysLeuArgMetLeuPhe                              505560                                                                        LysAspAspTyrProSerSerProProLysCysLysPheGluProPro                              65707580                                                                      LeuPheHisProAsnValTyrProSerGlyThrValCysLeuSerIle                              859095                                                                        LeuGluGluAspLysAspTrpArgProAlaIleThrIleLysGlnIle                              100105110                                                                     LeuLeuCysIleGlnGluLeuLeuAsnGluProAsnIleGlnAspPro                              115120125                                                                     AlaGlnAlaGluAlaTyrThrIleTyrCysGlnAsnArgValGluTyr                              130135140                                                                     GluLysArgValArgAlaGlnAlaLysLysPheAlaProSer                                    145150155                                                                     (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 152 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetSerThrProAlaArgArgArgLeuMetArgAspPheLysArgLeu                              151015                                                                        GlnGluAspProProValGlyValSerGlyAlaProSerGluAsnAsn                              202530                                                                        IleMetGlnTrpAsnAlaValIlePheGlyProGluGlyThrProPhe                              354045                                                                        GluAspGlyThrPheLysLeuLeuIleGluPheSerGluGluTyrPro                              505560                                                                        AsnLysProProThrValArgPheLeuSerLysMetPheHisProAsn                              65707580                                                                      ValTyrAlaAspGlySerIleCysLeuAspIleLeuGlnAsnArgTrp                              859095                                                                        SerProThrTyrAspValSerSerIleLeuThrSerIleGlnSerLeu                              100105110                                                                     LeuCysGluProAsnProAsnSerProAlaAsnSerGlnAlaAlaGln                              115120125                                                                     LeuTyrGlnGluAsnLysArgGluTyrGluLysArgValSerAlaIle                              130135140                                                                     ValGluGlnSerTrpAsnAspSer                                                      145150                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 152 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       MetSerThrProAlaArgArgArgLeuMetArgAspPheLysArgLeu                              151015                                                                        GlnGluAspProProValGlyValSerGlyAlaProSerGluAsnAsn                              202530                                                                        IleMetGlnTrpMetAlaValIlePheGlyProGluGlyThrProPhe                              354045                                                                        GluAspGlyThrPheLysLeuValIleGluPheSerGluGluTyrPro                              505560                                                                        AsnLysProProThrValArgPheLeuSerLysMetPheHisProAsn                              65707580                                                                      ValTyrAlaAspGlySerIleCysLeuAspIleLeuGlnAsnArgTrp                              859095                                                                        SerProThrTyrAspValSerSerIleLeuThrSerIleGlnSerLeu                              100105110                                                                     LeuAspGluProAsnProAsnSerProAlaAsnSerGlnAlaAlaGln                              115120125                                                                     LeuTyrGlnGluAsnLysArgGluTyrGluLysArgValSerAlaIle                              130135140                                                                     ValGluGlnSerTrpAsnAspSer                                                      145150                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 152 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetSerThrProAlaArgArgArgLeuMetArgAspPheLysArgLeu                              151015                                                                        GlnGluAspProProAlaGlyValSerGlyAlaProSerGluAsnAsn                              202530                                                                        IleMetValTrpAsnAlaValIlePheGlyProGluGlyThrProPhe                              354045                                                                        GlyAspGlyThrPheLysLeuThrIleGluPheThrGluGluTyrPro                              505560                                                                        AsnLysProProThrValArgPheValSerLysMetPheHisProAsn                              65707580                                                                      ValTyrAlaAspGlySerIleCysLeuAspIleLeuGlnAsnArgTrp                              859095                                                                        SerProThrTyrAspValSerSerIleLeuThrSerIleGlnSerLeu                              100105110                                                                     LeuAspGluProAsnProAsnSerProAlaAsnSerGlnAlaAlaGln                              115120125                                                                     LeuTyrGlnGluAsnLysArgGluTyrGluLysArgValSerAlaIle                              130135140                                                                     ValGluGlnSerTrpArgAspCys                                                      145150                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       MetSerThrProAlaArgArgArgLeuMetArgAspPheLysArgLeu                              151015                                                                        GlnGluAspProProThrGlyValSerGlyAlaProThrAspAsnAsn                              202530                                                                        IleMetIleTrpAsnAlaValIlePheGlyProHisAspThrProPhe                              354045                                                                        GluAspGlyThrPheLysLeuThrIleGluPheThrGluGluTyrPro                              505560                                                                        AsnLysProProThrValArgPheValSerLysValPheHisProAsn                              65707580                                                                      ValTyrAlaAspGlyGlyIleCysLeuAspIleLeuGlnAsnArgTrp                              859095                                                                        SerProArgTyrAspValSerAlaIleLeuThrSerIleGlnSerLeu                              100105110                                                                     LeuSerAspProAsnProAsnSerProAlaAsnSerThrAlaAlaGln                              115120125                                                                     LeuTyrLysGluAsnArgArgGluTyrGluLysArgValLysAlaCys                              130135140                                                                     ValGluGlnSerPheIleAsp                                                         145150                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      MetSerThrThrAlaArgArgArgLeuMetArgAspPheLysArgMet                              151015                                                                        GlnGlnAspProProAlaGlyValSerAlaSerProValSerAspAsn                              202530                                                                        ValMetLeuTrpAsnAlaValIleIleGlyProAlaAspThrProPhe                              354045                                                                        GluAspGlyThrPheLysLeuValLeuSerPheAspGluGlnTyrPro                              505560                                                                        AsnLysProProLeuValLysPheValSerThrMetPheHisProAsn                              65707580                                                                      ValTyrAlaAsnGlyGluLeuCysLeuAspIleLeuGlnAsnArgTrp                              859095                                                                        SerProThrTyrAspValAlaAlaIleLeuThrSerIleGlnSerLeu                              100105110                                                                     LeuAsnAspProAsnAsnAlaSerProAlaAsnAlaGluAlaAlaGln                              115120125                                                                     LeuHisArgGluAsnLysLysGluTyrValArgArgValArgLysThr                              130135140                                                                     ValGluAspSerTrpGluSer                                                         145150                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 172 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      MetSerThrProAlaArgArgArgLeuMetArgAspArgLysArgMet                              151015                                                                        LysGluAspAlaProProGlyValSerAlaSerProLeuProAspAsn                              202530                                                                        ValMetValTrpAsnAlaMetIleIleGlyProAlaAspThrProTyr                              354045                                                                        GluAspGlyThrPheArgLeuLeuLeuGluPheAspGluGluTyrPro                              505560                                                                        AsnLysProProHisValLysPheLeuSerGluMetPheHisProAsn                              65707580                                                                      ValTyrAlaAsnGlyGluIleCysLeuAspIleLeuGlnAsnArgTrp                              859095                                                                        ThrProThrTyrAspValAlaSerIleLeuThrSerIleGlnSerLeu                              100105110                                                                     PheAsnAspProAsnProAlaSerProAlaAsnValGluAlaAlaThr                              115120125                                                                     LeuPheLysAspHisLysSerGlnTyrValLysArgValLysGluThr                              130135140                                                                     ValGluLysSerTrpGluAspAspMetAspAspMetAspAspAspAsp                              145150155160                                                                  AspAspAspAspAspAspAspAspAspGluAlaAsp                                          165170                                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AAGTCGTCG9                                                                    (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GCAGCCATTG10                                                                  (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GGTACCGTCGACGCCGGCAAGCTTGCTGGATCCTGTACC39                                     GlyThrValAspAlaGlyLysLeuAlaGlySerCysThr                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GlyThrValAspAlaGlyLysLeuAlaGlySerCysThr                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 430 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      ProSerLysTrpGlyAspArgProValGlyGlyGlyProSerAlaGly                              151015                                                                        ProValGlnGlyLeuGlnArgLeuLeuGlnAlaLysSerProGlyGlu                              202530                                                                        LeuLeuArgTrpLeuGlyArgAsnProSerLysValArgAlaHisHis                              354045                                                                        TyrSerValAlaLeuArgArgLeuGlyGlnLeuLeuGlySerArgPro                              505560                                                                        ArgProProProValGluGlnValThrLeuGlnAspLeuSerGlnLeu                              65707580                                                                      IleIleArgAsnCysProSerPheAspIleHisThrIleHisValCys                              859095                                                                        LeuHisLeuAlaValLeuLeuGlyPheProSerAspGlyProLeuVal                              100105110                                                                     CysAlaLeuGluGlnGluArgArgLeuArgLeuProProLysProPro                              115120125                                                                     ProProLeuGlnProLeuLeuArgGlyGlyGlnGlyLeuGluAlaAla                              130135140                                                                     LeuSerCysProArgPheLeuArgTyrProArgGlnHisLeuIleSer                              145150155160                                                                  SerLeuAlaGluAlaArgProGluGluLeuThrProHisValMetVal                              165170175                                                                     LeuLeuAlaGlnHisLeuAlaArgHisArgLeuArgGluProGlnLeu                              180185190                                                                     LeuGluAlaIleAlaHisPheLeuValValGlnGluThrGlnLeuSer                              195200205                                                                     SerLysValValGlnLysLeuValLeuProPheGlyArgLeuAsnTyr                              210215220                                                                     LeuProLeuGluGlnGlnPheMetProCysLeuGluArgIleLeuAla                              225230235240                                                                  ArgGluAlaGlyValAlaProLeuAlaThrValAsnIleLeuMetSer                              245250255                                                                     LeuCysGlnLeuArgCysLeuProPheArgAlaLeuHisPheValHis                              260265270                                                                     SerProGlyPheIleAsnTyrIleSerGlyThrProHisAlaLeuIle                              275280285                                                                     ValArgArgThrLeuSerLeuLeuAspThrAlaValGluLeuGluLeu                              290295300                                                                     ProGlyTyrArgGlyProArgLeuProArgArgGlnGlnValProIle                              305310315320                                                                  PheProGlnProLeuIleThrAspArgAlaArgCysLysTyrSerHis                              325330335                                                                     LysAspIleValAlaGluGlyLeuArgGlnLeuLeuGlyGluGluLys                              340345350                                                                     TyrArgGlnAspLeuThrValProProGlyTyrCysThrAspPheLeu                              355360365                                                                     LeuCysAlaSerSerSerGlyAlaValLeuProValArgThrGlnAsp                              370375380                                                                     ProPheLeuProTyrProProArgSerCysProGlnGlyGlnAlaAla                              385390395400                                                                  SerSerAlaThrThrArgAspProAlaGlnArgValValLeuValLeu                              405410415                                                                     ArgGluArgTrpHisPheSerArgAspGlyArgValLeuLeu                                    420425430                                                                     (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 382 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ValAlaValThrAsnIleGlyAlaGlySerAspGlyGlyThrAlaVal                              151015                                                                        ValAlaPheGlyGlyThrProArgArgGlyGlyGluGlyAspProVal                              202530                                                                        GlyProAlaGluPheValSerAspAspArgSerSerAspSerAspSer                              354045                                                                        AspAspSerGluAspThrAspSerGluThrIleSerHisAlaSerSer                              505560                                                                        AspValSerGlyGlyAlaThrTyrAspAspAlaLeuAspSerAspSer                              65707580                                                                      SerSerAspAspSerLeuGlnIleAspGlyProValCysArgProTrp                              859095                                                                        SerAsnAspThrAlaProLeuAspValCysProGlyThrProGlyPro                              100105110                                                                     GlyAlaAspAlaGlyGlyProSerAlaValAspProHisAlaProThr                              115120125                                                                     ProGluAlaGlyAlaGlyLeuAlaAlaAspProAlaValAlaArgAsp                              130135140                                                                     AspAlaGluGlyLeuSerAspProArgProArgLeuGlyThrGlyThr                              145150155160                                                                  AlaTyrProValProLeuGluLeuThrProGluAsnAlaGluAlaVal                              165170175                                                                     AlaArgPheLeuGlyAspAlaValAsnArgGluProAlaLeuMetLeu                              180185190                                                                     GluTyrPheCysArgCysAlaArgGluGluThrLysArgValProPro                              195200205                                                                     ArgThrPheGlySerProProArgLeuThrGluAspAspPheGlyLeu                              210215220                                                                     LeuAsnTyrAlaLeuValGluMetGlnArgLeuCysLeuAspValPro                              225230235240                                                                  ProValProProAsnAlaTyrMetProTyrTyrLeuArgGluTyrVal                              245250255                                                                     ThrArgLeuValAsnGlyPheLysProLeuValSerArgSerAlaArg                              260265270                                                                     LeuTyrArgIleLeuGlyValLeuValHisLeuArgIleArgThrArg                              275280285                                                                     GluAlaSerPheGluGluTrpLeuArgSerLysGluValAlaLeuAsp                              290295300                                                                     PheGlyLeuThrGluArgLeuArgGluHisGluAlaGlnLeuValIle                              305310315320                                                                  LeuAlaGlnAlaLeuAspHisTyrAspCysLeuIleHisSerThrPro                              325330335                                                                     HisThrLeuValGluArgGlyLeuGlnSerAlaLeuLysTyrGluGlu                              340345350                                                                     PheTyrLeuLysArgPheGlyGlyHisTyrMetGluSerValPheGln                              355360365                                                                     MetTyrThrArgIleAlaGlyPheLeuAlaCysArgAlaThr                                    370375380                                                                     (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 395 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      PheValAlaIleSerAsnValAlaAlaGlyGlyAsnGlyArgThrAla                              151015                                                                        ValValAlaLeuGlyGlyThrSerGlyAlaArgGlyGlyAlaGluLys                              202530                                                                        AspValGlyAlaAlaGluSerTrpSerAspGlyProSerSerAspSer                              354045                                                                        GluThrGluAspSerAspSerSerAspGluAspThrGlySerGlySer                              505560                                                                        GluThrLeuSerArgSerSerSerIleTrpAlaAlaGlyAlaThrAsp                              65707580                                                                      AspAspAspSerAspSerAspSerArgSerAspAspSerValGlnPro                              859095                                                                        AspValValValArgArgArgTrpSerAspGlyProAlaProValAla                              100105110                                                                     PheProLysProArgArgProGlyAspSerProGlyAsnProGlyLeu                              115120125                                                                     GlyAlaGlyThrGlyProGlySerAlaThrAspProArgAlaSerAla                              130135140                                                                     AspSerAspSerAlaAlaHisAlaAlaAlaProGlnAlaAspValAla                              145150155160                                                                  ProValLeuAspSerGlnProThrValGlyThrAspProGlyTyrPro                              165170175                                                                     ValProLeuGluLeuThrProGluAsnAlaGluAlaValAlaArgPhe                              180185190                                                                     LeuGlyAspAlaValAspArgGluProAlaLeuMetLeuGluTyrPhe                              195200205                                                                     CysArgCysAlaArgGluGluSerLysArgValProProArgThrPhe                              210215220                                                                     GlySerAlaProArgLeuThrGluAspAspPheGlyLeuLeuAsnThr                              225230235240                                                                  AlaLeuAlaGluMetArgArgLeuCysLeuAspLeuProProValPro                              245250255                                                                     ProAsnAlaTyrThrProTyrHisLeuArgGluTyrAlaThrArgLeu                              260265270                                                                     ValAsnGlyPheLysProLeuValArgArgSerAlaArgLeuTyrArg                              275280285                                                                     IleLeuGlyIleLeuValHisLeuArgIleArgThrArgGluAlaSer                              290295300                                                                     PheGluGluTrpMetArgSerLysGluValAspLeuAspProGlyLeu                              305310315320                                                                  ThrGluArgLeuArgGluHisGluAlaGlnLeuMetIleLeuAlaGln                              325330335                                                                     AlaLeuAsnProTyrAspCysLeuIleHisSerThrProAsnThrLeu                              340345350                                                                     ValGluArgGlyLeuGlnSerAlaLeuLysTyrGluGluHisTyrLeu                              355360365                                                                     LysArgHisGlyGlyHisTyrMetGluSerValHisGlnMetTyrThr                              370375380                                                                     ArgIleAlaGlyProLeuAlaCysArgAlaThr                                             385390395                                                                     (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 282 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      MetGluAsnTyrGlnLysValGluLysIleGlyGluGlyThrTyrGly                              151015                                                                        ValValTyrLysAlaArgHisLysLeuSerGlyArgIleValAlaMet                              202530                                                                        LysLysIleArgLeuGluAspGluSerGluGlyValProSerThrAla                              354045                                                                        IleArgGluIleSerLeuLeuLysGluValAsnAspGluAsnAsnArg                              505560                                                                        SerAsnCysValArgLeuLeuAspIleLeuHisAlaGluSerLysLeu                              65707580                                                                      TyrLeuValPheGluPheLeuAspMetLysLeuLysLysTyrMetAsp                              859095                                                                        ArgIleSerPheThrGlyAlaThrSerLeuAspProArgLeuValGln                              100105110                                                                     LysPheThrTyrGlnLeuValAsnGlyValAsnPheCysHisSerArg                              115120125                                                                     ArgIleIleHisArgAspLeuLysProGlnAsnLeuLeuIleAspLys                              130135140                                                                     GluGlyAsnLeuLysLeuAlaAspPheGlyLeuAlaArgSerPheGly                              145150155160                                                                  ValProLeuArgAsnTyrThrHisGluIleValThrLeuTrpTyrArg                              165170175                                                                     AlaProGluValLeuLeuGlySerArgHisTyrSerThrGlyValAsp                              180185190                                                                     IleTrpSerValGlyCysIlePheAlaGluMetIleArgArgSerPro                              195200205                                                                     LeuPheProGlyAspSerGluIleAspGluIlePheLysIlePheGln                              210215220                                                                     ValLeuGlyThrProAsnGluGluValTrpProGlyValThrLeuLeu                              225230235240                                                                  GlnAspTyrLysSerThrPheProArgTrpLysArgMetAspLeuTyr                              245250255                                                                     HisLysValValProAsnGlyGluGluAspAlaIleGluLeuLeuSer                              260265270                                                                     AlaMetLeuValTyrAspProAlaHisArg                                                275280                                                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 274 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      MetGluAsnPheGlnLysValGluLysIleGlyGluGlyThrTyrGly                              151015                                                                        ValValTyrLysAlaArgAsnLysLeuThrGlyGluValValAlaLeu                              202530                                                                        LysLysIleArgLeuAspThrGluThrGluGlyValProSerThrAla                              354045                                                                        IleArgGluIleSerLeuLeuLysGluLeuAsnHisProAsnIleVal                              505560                                                                        LysLeuLeuAspValIleHisThrGluAsnLysLeuTyrLeuValPhe                              65707580                                                                      GluPheLeuHisGlnAspLeuLysLysPheMetAspAlaSerAlaLeu                              859095                                                                        ThrGlyIleProLeuProLeuIleLysSerTyrLeuPheGlnLeuLeu                              100105110                                                                     GlnGlyLeuAlaArgCysHisSerHisArgValLeuHisArgAspLeu                              115120125                                                                     LysProGlnAsnLeuLeuIleAsnThrGluGlyAlaIleLysLeuAla                              130135140                                                                     AspPheGlyLeuAlaArgAlaPheGlyValProValArgThrTyrThr                              145150155160                                                                  HisGluValValThrLeuTrpTyrArgAlaProGluIleLeuLeuGly                              165170175                                                                     SerLysTyrTyrSerThrAlaValLysIleTrpSerLeuGlyCysIle                              180185190                                                                     PheAlaGluMetValThrArgArgAlaLeuPheProGlyAspSerGlu                              195200205                                                                     IleAspGlnLeuPheArgIlePheArgThrLeuGlyThrProAspGlu                              210215220                                                                     ValValTrpProGlyValThrSerMetProAspTyrLysProSerPhe                              225230235240                                                                  ProLysTrpAlaArgGlnAspPheSerLysValValProProLeuAsp                              245250255                                                                     GluAspGlyArgSerLeuLeuSerGlnMetLeuHisTyrAspProAsn                              260265270                                                                     LysArg                                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 244 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GluSerLeuArgLeuGluValLysLeuGlyGlnGlyCysArgGlyGlu                              151015                                                                        ValTrpMetGlyIleTrpAsnGlyThrThrArgValAlaIleLysThr                              202530                                                                        LeuLysProGlyThrMetSerProGluAlaPheLeuGlnGluAlaGln                              354045                                                                        ValMetLysLysLeuArgHisGluLysLeuValGlnLeuTyrAlaVal                              505560                                                                        ValSerGluGluProIleTyrIleValThrGluTyrMetSerLysGly                              65707580                                                                      SerLeuLeuAspPheLeuLysGlyGluThrGlyLysTyrLeuArgLeu                              859095                                                                        ProGlnLeuValAspMetAlaAlaGlnIleAlaSerGlyMetAlaTyr                              100105110                                                                     ValGluArgMetAsnTyrValHisArgAspLeuArgAlaAlaAsnIle                              115120125                                                                     LeuValGlyGluAsnLeuValCysLysValAlaAspPheGlyLeuAla                              130135140                                                                     ArgLeuIleGluAspAsnGluTyrThrAlaArgGlnGlyAlaLysPhe                              145150155160                                                                  ProIleLysTrpThrAlaProGluAlaAlaLeuTyrGlyArgPheThr                              165170175                                                                     IleLysSerAspValTrpSerArgGlyIleLeuLeuThrGluLeuThr                              180185190                                                                     ThrLysGlyArgValProTyrProGlyMetValAsnArgGluValLeu                              195200205                                                                     AspGlnValGluArgGlyTyrArgMetProCysProProGluProGlu                              210215220                                                                     SerLeuHisAspLeuMetCysGlnCysTrpArgLysGluProGluGlu                              225230235240                                                                  ArgProThrPhe                                                                  __________________________________________________________________________

What is claimed is:
 1. A substantially pure polypeptide that has anamino acid sequence that is SEQ ID NO:2.
 2. A substantially purepolypeptide that has an amino acid sequence that is SEQ ID NO:4.